PB2386: ASSESSMENT OF NANOPORE LONG-READ WHOLE-GENOME SEQUENCING FOR THE DETECTION OF LARGE CHROMOSOMAL STRUCTURAL VARIANTS IN MANTLE CELL LYMPHOMA
Authors: Marcus Hansen, Oriane Cédile, Marie L.G. Kjeldsen, Mads Thomassen, Birgitte Preiss, Niels Abildgaard, Charlotte Guldborg Nyvold
Journal: HemaSphere (2023)

Topic: 20. Lymphoma Biology & Translational Research Background: Detection of t(11;14)(q13;q32), a hallmark of mantle cell lymphoma (MCL), remains challenging when relying on next-generation sequencing (NGS) in contrast to fluorescence in-situ hybridization. First, the breakpoint locations vary from patient to patient. Secondly, the translocation does not result in a chimeric fusion protein and can, thus, not be detected by RNA sequencing or PCR of cDNA. Furthermore, the breakpoint does not alter the gene or coding region of CCND1 but deregulates transcriptional regulation kilobases upstream. Aims: Because of this challenge, the study evaluated the detection and reproducibility of extensive copy-number alterations and chromosomal translocations using long-read Oxford Nanopore Technologies (ON). Methods: DNA from GRANTA-519 (German Collection of Microorganisms and Cell Cultures) and donor was extracted with the AllPrep DNA/RNA mini kit (Qiagen). The ON long-read whole-genome sequencing (WGS) library was prepared from 1 µg DNA using the Ligation Sequencing Kit, SQK-LSK110, ON) with enrichment of long fragments. Replicate libraries were each loaded onto primed R9.4.1 flow cells (ON) and sequenced on MinION Mk1C. Raw sequencing reads were aligned to GRCh38 reference using Minimap2. WGS of Granta-519 and donor on NovaSeq 6000 System (Illumina) was performed using DNA PCR-Free Prep, and alignment with Burrows-Wheeler Aligner. Coverage profiles were obtained through BEDTools multicov and QDNAseq with a fixed resolution of 100 Kb. Coverage profiles were normalized to the median number of reads and smoothened for noise reduction (gaussian and median filter). Selected regions were confirmed by qPCR using TaqMan Copy Number Assays CDKN2A, CDKN2B, BCL6, TP53, CCNL1, TERT) using the QuantStudio 12K Flex System and CopyCaller Software (Applied Biosystems). Results: The effective yield from ON sequencing was 14.9–19.2·109 mapped bases, i.e., 5–6x coverage and 49–50x for Illumina. Based on sequencing of the MCL cell line Granta-519, we demonstrate almost 99% reproducibility of copy-number at a 100 Kb resolution between ON replicates, with 98% overlap to Illumina (2,623 Mb). Collectively, the performance of copy-number calling from 1.5–7.5 million long reads, on average, was comparable to Illumina samples attaining nearly 1 billion reads, albeit with reduced noise in the latter. Expectedly, the ON long-read resolution of the canonical translocation t(11;14)(q13;q32) (9x coverage) was superior to the Illumina sequencing (5x coverage) with a mean local alignment similarity for ON of 89% to the already published CCND1/IGH junction, spanning up to 69 Kb in a single read. Summary/Conclusion: Although ON sequencing has a ten-fold higher base-calling error rate than Illumina, subsequent analysis of the Granta-519 t(11;14) breakpoint matched the published CCND1/IgH JH4 translocation breakpoint (GenBank Y11645). The robustness of ON sequencing makes a central role in future cytogenomics possible, although the combined strength of short and long-read sequencing is apparent. As the coverage for the short-read sequencing here was several-fold higher than obtained with long-read sequencing, the two technologies may be comparable for resolving chromosomal copies. Keywords: Genomics, Cytogenetics, Molecular cytogenetics, Cytogenetic abnormalities